PROJECT SUMMARY Natural Killer (NK) cells are highly relevant in the setting of hematopoietic stem cell transplantation (HCT), where their activity can drastically reduce the risk of relapse in leukemia patients. Despite advances in donor selection to minimize transplant complications, leukemia relapse remains one of the most devastating causes of transplant failure. There is a general lack of knowledge of how NK cell populations are educated for effector function, leukemia recognition and clearance. While it is known that interaction between HLA class I molecules and the inhibitory killer Ig-like receptors (KIR) are important for NK education or ?licensing? for effector function, the orientation of their interaction, the factors that contribute to the strength of their interaction, and the relative contributions of donor and recipient HLA are not known. A major impediment to fundamental human NK studies has been the lack of in vivo models that faithfully recapitulate NK education. A humanized NOD-RAG2IL2R?c-/- mouse strain that exhibits HLA-B*27:05 successfully permits engraftment of mature NK cells and development of functional human NK cells from CD34+ stem cells. Preliminary investigations reveal that in vivo environments expressing cognate HLA educate developing and transferred KIR-expressing NK cells and enhance their long-term survival. In parallel, FRET and RNAi technology reveals that cis-interaction between HLA and KIR intrinsic to the cell also facilitates NK education. Evidence that HLA molecules can be transferred onto NK cells from adjacent cells provokes investigation of trogocytosis as a mechanism by which trans HLA may permit education via cis ligation of KIR. Restriction of HLA expression to the hematopoietic or stromal compartments in bone marrow chimeras will help define the relative contributions of donor and recipient HLA to NK licensing and persistence. Receptor- ligand pairs known to bind in trans also interact in cis, and further clarification is required to understand how these interactions influence NK function. Competition between cis-trans binding will test the hypothesis that cis interactions facilitate NK responsiveness by shielding KIR from ligation of trans HLA. NK cells with and without non-native HLA expression will be functionally tested to better understand how trogocytosis contributes to NK education after continuous or interrupted exposure to cognate HLA. Finally, experimental findings from the humanized mouse model and in vitro studies will be verified in a fully human system using NK cells obtained from patients who have received an HLA-mismatched umbilical cord blood transplant. These goals will advance our understanding of fundamental molecular requirements for NK education and acquisition of effector function. A deeper understanding of licensing by HLA provided in cis and trans will inform donor selection in hematopoietic cell transplantation and adoptive NK cell therapies for the treatment of cancer.